Efficient methods for fast, producible, C-Phycocyanin from Thermosynechococcus elongatus

Journal of Research in Biology ISSN No: Print:2231 - 6280; Online: 2231 - 6299. An International Scientific Research Journal Original Research Efficient methods for fast, producible, C-Phycocyanin from Thermosynechococcus elongatusJournal of Research in Biology Authors: ABSTRACT: El-Mohsnawy Eithar. This article describes different protocols that enhance the extraction, isolation Institution: and purification of phycocyanin from the cyanobacterium, Thermosynechococcus Botany Department, Faculty elongatus as well as absorbance and fluorescence spectral characterization. A combination of Science, Damanhour of enzymatic degradation by Lysozyme followed by high pressure showed a mild cell wall University, 22713, Egypt. destruction except for the composition of thylakoid membrane compared with glass beads. The use of ammonium sulfate precipitation as the first purification step exhibited high efficiency in removing most of the protein contamination. The best purified phycocyanin was obtained after using the second purification step that could be ion exchange chromatography or sucrose gradient. Unexpected results that were not used earlier were obtained by sucrose gradient, where a large amount of highly pure phycocyanin was assembled compared with published methods. An evaluation of C-phycocyanin throughout the series steps of isolation and purification was achieved by using absorbance and 77K fluorescence spectral analysis. Besides a spectroscopical evaluation, SDS-PAGE, productivity, and A620/A280 values pointed to the purity and structural preservation of a purified complex. Compared with published methods, the existing method not only reduces purification time but also enhances the productivity of phycocyanin in its native structure. The optimization of each purification step presented different purified phycocyanin levels; hence, it could be used not only by microbiologists but also by other researchers such as physicians and industrial applicants. In addition, this method could be used as a model for all cyanobacterial species and could be also used for Rhodophytes with some modifications. Corresponding author: Keywords: El-Mohsnawy Eithar. A620/A280 value, C-PC purification, C-Phycocyanin, Cyanobacteria, Fluorescence Spectra, IEC, Phycobilines, Sucrose Gradient, Thermosynechococcus elongatus. Abbreviations A620/A280: Absorbance at 620 and 280 nm; Amm Sulf. ppt: Ammonium sulfate precipitate; APC: Allophycocyanin; MCF-7: Michigan Cancer Founda,on-7, referring to the ins;tute in Detroit where the cell line was established in 1973; OD: Op;cal density., PBP: Phycobilliprotein; PC (C-PC): Phycocyanin (phycocyanin from cyanobacteria); T. elongatus: Thermosynechococcus elongates; IEC: Ion exchange column. Web Address: Article Citation: http://jresearchbiology.com/ El-Mohsnawy Eithar. Efficient methods for fast, producible, C-Phycocyanin from Thermosynechococcus elongates. documents/RA0419.pdf. Journal of Research in Biology (2014) 3(8): 1132-1146 Dates: Received: 24 Jan 2014 Accepted: 05 Feb 2014 Published: 10 Feb 2014 Journal of Research in Biology © The author. This article is governed by the Creative Commons Attribution License (http:// An International creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. Scientific Research Journal 1132-1146 | JRB | 2014 | Vol 3 | No 8 Published by Redolence Academic Services www.jresearchbiology.com

El-Mohsnawy Eithar, 2014INTRODUCTION One function of PC is energy absorbance which Blue green are one of oldest prokaryotic fossils is transferred by non-radiative transfer into APC and consequently into chlorophyll a, with an efficiency(Schopf 2000) that have been known on the earth formore than 3.5 billion years. The traditional name ‘blue- approaching 100%. In the absence or blocked thegreen algae’ for Cyanophyceae is due to the presence of photosynthetic reaction center (RC), the PBPs arephycocyanin, allophycocyanin, and phycoerythrin, which strongly fluorescent.mask the chlorophyll pigmentation. Most cyanobacteria C-phycocyanin is composed of two subunits: theare found in fresh water, whereas others are found in α-chain with one phycocyanobilin and the β-chain withmarines, in damp soil, or even in temporarily moistened two phycocyanobilins (Troxler et al., 1981; Stec et al.,rocks in deserts as well as in hot springs such as 1999; Adir et al., 2001; Contreras-Martel et al., 2007). InThermosynechococcus elongatus. T. elongatus is between, there are large amino-acid sequenceconsidered a thermophilic obligate photoautotrophic similarities. The αβ subunits aggregate into α3 β3 trimersorganism that contains chlorophyll a, carotenoids, and and further into disc-shaped α6 β6 hexamers, thephycobilins. For this reason, it has usually been used as a functional unit of C-PC (Stec et al., 1999; Adir et al.,model organism for the study of photosynthesis; such as, 2001; Contreras-Martel et al., 2007).X-ray structure of PSI and PSII (Sonoike and Katoh Nowadays, Phycocyanin receives a lot of1989; Zouni et al., 2001; Jordan et al., 2001; and Katoh attention due to its potential in medical andet al., 2001). pharmaceutical treatments as well as in food industries. Its antioxidant protection of DNA has been demonstrated In addition, Thermosynechococcus elongatus has by (Pleonsil and Suwanwong, 2013). It also promotesbeen postulated as the model organism of choice forstructural studies. X-ray of photosystem I are studied by PC12 cell survival, modulates immune and inflammatoryJordan et al., 2001 and photosystem II are studied by genes and oxidative stress markers in acute cerebralFerreira et al., 2004 and Loll et al., 2005. A crystal hypoperfusion in rats (Marín-Prida et al., 2013), preventsstructure of the cytochrome b6f complex has been hypertension and low serum adiponectin level in a ratdetermined from another thermophilic cyanobacterium, model of metabolic syndrome (Ichimura et al., 2013),Mastigocladus laminosus (Kurisu et al., 2003). exhibits an antioxidant and in vitro antiproliferative activity (Thangam et al., 2013), and involves an The thylakoid membrane of apoptotic mechanism of MCF-7 breast cells either in vivoThermosynechococcus elongatus attached to external or in vitro induced by photodynamic therapy withlight-harvesting structure known as the phycobilisome C-phycocyanin (Li et al., 2010).(PBS; reviewed by Adir 2005), which acts as a light-harvesting system for PSII and, to some extent, for PSI For these reasons, a lot of attention is directed(Rögner et al., 1996). The Synechococcus elongatus toward improving the purification of phycocyanin fromphycobilisome consists of allophycocyanin (APC) and several cyanobacterial organisms. The purification ofC-phycocyanin (C-PC), along with the linker proteins C-phycocyanin from Spirulina platensis has been(Adir, 2005). The bilin pigments are open-chained reported by Bhaskar et al., (2005); from Anacystistetrapyrroles that are covalently bound to seven or more nidulans (Gupta and. Sainis 2010); and in aqueousproteins. These chromophores are composed of the phytoplankton by Lawrenz et al., 2011.cyclic iron (heme) tetrapyrrole (Frankenberg and Although all these represented evaluations wereLagarias 2003; Frankenberg et al., 2003). based on the ratio of A620/A280, which is suggested by1133 Journal of Research in Biology (2014) 3(8): 1132-1146

El-Mohsnawy Eithar, 2014Bryant et al., (1979) and Boussiba and Richmond (1979), MES, 10 mM Magnesium chloride, and 10 mM Calciumthis ratio does not save an optimum image of the Chloride and 0.2 % (w/v) Lysozyme). Stirring waspresence of other impurities such as APC with applied at 37 °C for 30 minutes in the dark condition. InC-phycocyanin, where the existence of APC does not the first protocol, the cell wall was disrupted by applyingstrongly disturb this ratio. Purity ratios varied among 2000 psi pressure using Parr bomb at at 4°C for 20publications: 4.3 (Minkova et al., 2003), 3.64 (Niu et al., minutes (El-Mohsnawy et al., 2010). However, in the2007), 4.05 (Patil and Raghavarao 2007), 4.72 (Gupta second protocol was done according to Kubota et al.,and Sainis 2010), and more than four (Pleonsil and 2010, where T. elongatus cells were mixed with an equalSuwanwong 2013). volume of glass beads (0.5 mm of Glass Beads, Soda Lime, BioSpec Products), and then, the cells were This article displays the simple, fast, and exposed to 18 disrupted cell cycles (10s ec glass beadseffective protocol by which large scales of PC were break and 2min 50sec pause) on a vortex mixer (BSPpurified. Bead-Beater 1107900, BioSpec Products).MATERIAL AND METHODS Phycocyanin crude extract was collected by suspending the thylakoid membrane with HEPES bufferCulturing and assembly of T. elongatus at pH 7.5 (20mM HEPES, 10mM MgCl2, 10 mMCaCl2, Thermosynechococcus elongatus cells were and 0.4 M mannitol) or with HEPES buffer at pH 7.5 containing 0.03% ß-DM and centrifugation at 3000 g at 4cultivated in BG-11 medium at 50 °C with a stream of °C for 10 min. The supernatant was collected, and pellets5% (v/v) CO2 in air (according to Rippka et al., 1979). were exposed to an additional extraction step using theCells were grown in Polyamide flasks (2.5-L). 200-ml same buffer and centrifugation conditions. By usingpreculture cells were used for an inoculation of 2 L glass bead disruption, an additional isolation step was notculture. The used white light was provided at about 100 required.µE*m-2*s-1. After incubation period, the cells wereharvested in the exponential growth phase. The optical Purification stepsdensity at 750 nm was 2.5 - 3. First purification step: Cells were sedimented by centrifugation at 2000 This step was preceded using two sequences ofg for 15 minutes (GSA-Rotor, Sorvall). The supernatantwas removed. Cells in the pellet were washed once with ammonium sulfate precipitation steps. AmmoniumMES buffer (20mM MES, 10 mM Magnesium chloride, sulfate salts were added to the crude extract in HEPESand 10 mM Calcium Chloride) and then re-centrifuged at buffer till it reached 20 %, was stirred at 4°C for 30the same speed and conditions. minutes followed by centrifugation of 6000 g at 4 °C for 15 min (Beckman -JA-14 Rotor). The pellets wereExtraction of phycocyanin discarded. Additional ammonium sulfate salts were The extraction of phycocyanin crude extract was added to the supernatant till they reached 50 % saturation and were stirred at 4°C for 60 minutes. Centrifugation ofperformed in two steps. The first step was cell wall 12000 g at 4 °C for 30 min (Beckman -JA-14 Rotor) wasdestruction, and the second step was isolation of used to sediment partial purified phycocyaninphycocyanin from the thylakoid membrane. Two (El-Mohsnawy, 2013).destruction techniques were applied. In both techniques,collected T. elongatus cells were suspended in 100 ml ofMES containing Lysozyme buffer at pH 6.5 (20mMJournal of Research in Biology (2014) 3(8): 1132-1146 1134

El-Mohsnawy Eithar, 2014Second purification step: the current was reduced to 60 mA until the samples Pellets were dissolved in HEPES buffer at pH 7.5 reached the edge of the gel. After electrophoresis, SDS- PAGE was fixed by incubation in a mixture of 50 %(20mM HEPES, 10mM MgCl2, 6mM CaCl2, and 0.4 M methanol and 10% acetic acid for 20 min. The gel wasagainst HEPES buffer at pH 7.5 (20mM HEPES, 10mM stained with Coomassie Brilliant Blue reagent (0.2% (w/MgCl2, 10mMCaCl2, and 0.4 M mannitol) for 6 hours v), Coomassie Brilliant Blue R, 40% (v/v) methanol, andbefore loading to IEC (POROS HQ/M). 7 % (v/v) acetic acid) for an additional 20 min. The gel was destained by immersing the gel in a mixture of 30 %Sucrose gradient (v/v) methanol and 10 % (v/v) acetic acid for 8–12 hours. Sucrose gradient was prepared by dissolving Absorption spectral analysis20 % (w/v) sucrose in HEPES buffer at pH 7.5 (20mM 1 ml of crude or purified phycocyanin complexesHEPES, 10mM MgCl2, and 10 mMCaCl2). 12 ml ofsucrose solution was poured into each centrifuge tube was diluted in buffer (20 mM HEPES, pH 7.5, 10 mM(SW40-Rotor ultracentrifuge, Beckman) followed by MgCl2, 10 mM CaCl2, and 0.5 M mannitol) till itfreezing and slowly thawing overnight at 10°C. 100 µl of reached a maximum OD620 nm of 0.2–0.8 beforeOD620 nm 6 suspensions were slowly dropped onto the measuring the absorption spectra from 250 to 750 nm.top of sucrose gradients. After centrifugation at 36000 While thylakoid pellets were diluted to OD680 nm of 1.2-rpm for about 12 hours at 4°C (SW40-Rotor 2. Two spectrophotometers are used according to theultracentrifuge, Beckman), two identical bands were purpose of measurements. For fast evaluation of thedetected. The lower band (phycocyanin) was collected efficiency of each purification step, 2 µl of sample wasfor further investigation. used (NanoDrop ND-1000 Spectrophotometer). 500 µl samples were used in case of Shimadzu UV-2450 orIon Exchange Chromatography (IEC) Beckman Du7400. Phycocyanin concentration was POROS HQ/M column was used as IEC for the estimated according to an equation suggested by Bennett and Bogorad 1973; Bryant et al. 1979:second purification step. The column was equilibrated by8 CV of IEC equilibration buffer (20 mM MES, pH 6.5, PC (mg.ml) = {A620 – (0.7*A650)}/ 7.3810mM MgCl2, and 10 mMCaCl2) before loading thephycocyanin suspension. After loading the samples, Fluorescence emission spectra at 77 Kwashing occurred for 5 CV. The gradient from 0 to 200 Fluorescence emission spectra were performed inmM MgSO4 with a step at 35 mM that was carried outfor the elution of purified C-phycocyanin complex. an SLM-AMINCO Bauman, Series 2 LuminescencePurified phycocyanin was eluted at 23 mM MgSO4. spectrometer (Schlodder et al., 2007). PhycocyaninPurified phycocyanin was concentrated by centrifugation complex was diluted to OD620 nm 0.05 buffer containingat 3000 r/min for 40 min at 4°C using an Amicon 10,000 20 mM HEPES, pH 7.5, 10 mM MgCl2, 10 mM CaCl2,Dalton weight cut-off. and 60 % glycerol. The diluted sample was frozen to 77 K by gradual immersion in liquid nitrogen. 580 nm ofSDS-polyacrylamide gel electrophoresis (SDS-PAGE) actinic light was used for excitation. Fluorescence SDS-PAGE was performed according to emission spectra were monitored in the range from 600 to 800 nm with a step size of 1 nm and a bandpass filterSchägger and Von Jagow (1987). Briefly, 6 µl of of 4 nm.phycocyanin (OD620 nm 3) was mixed with samplebuffer. Then, the mixture was injected into SDS-PAGE(12% Acrylamide). The electrophoresis was carried outby applying a current of 100 mA for 30 min, and then,1135 Journal of Research in Biology (2014) 3(8): 1132-1146

El-Mohsnawy Eithar, 2014RESULTS: glass-bead destruction yielded a large amount of The purification of phycocyanin from allophycocyanin which has a maximum absorbance at 650 nm, in addition to small peaks at 680 nm for PSI andT. elongatus cells was achieved via several steps, so the 673 nm for PSII that also have a maximum absorbanceoptimization of each step was required to enhance the of nearly 440 nm. The absorption spectrum at 650 nmproductivity as well as the purity of phycocyanin. The proves the contamination of C-phycocyanin by a largescheme shown in Figure 1 illustrates the summary steps amount of allophycocyanin, whereas the absorbance atof extraction and purification of phycocyanin. 280 nm proves the presence of an additional large amount of non-colored proteins. Extraction by HEPESCell destruction and extraction of crude extract. buffer showed a small shoulder at 650 nm, compared Two different techniques have been used for cell with the same buffer containing ß-DM. A remarkable peak at 440 nm and small shoulders were observed atdestruction: combination of 0.2 % Lysozyme with 650 nm and 680 nm in case of HEPES buffer containingpressure (2000 psi) or combination of 0.2 % Lysozyme ß-DM, which confirmed the contamination with PS (Iwith glass-beads vortex. 0.2 % Lysozyme with pressure and II) complexes. It should be pointed out that the high(2000 psi) exhibited mild destruction of the cell wall absorbance value of HEPES buffer containing ß-DMwhile keeping the thylakoid membrane in its native compared with other treatments may reflect the ability ofstructure, even the attached phycobilisomes. After cell ß-DM to dissolve large amounts of protein which do notdestruction, the crude extract was isolated using HEPES have absorption spectra in visible regions. However, high(pH 7.5) buffer or HEPES (pH 7.5) containing 0.03 % ß- contamination of crude extract by allophycocyanin inDM. Both crude extracts exhibited different case of using glass beads did not exhibit a big differencespectroscopical behavior. On the other hand, glass beads in A620/A680 value (Table 1) compared with HEPESdestroyed the cell wall and thylakoid membrane, so extraction.centrifugation led to sedimentation of the largestphotosynthetic complexes. Figure 2a, b shows the This is regarding the close of absorption spectraabsorbance comparison between Lysozyme + HEPES, between allophycocyanin and phycocyanin (650 andLysozyme + HEPES containing 0.03 % ß-DM, andextraction by glass beads. It is obvious that the use ofTable 1 a: Summary of purity of phycocyanin (expressed as A620/A280 ratio), productivity (expressed as percent to crude extracts), and required periods for each step. Step A620/A280 ratio Productivity % Estimation TimeCrude HEPES 1.02909 ± 0.08229 100 30.0 min.Crude ß-DM 0.26732 ± 0.05131 100 30.0 min.Crude Beads 1.09185 ± 0.07352 100 30.0 min.After Amm Sulf. ppt 3.49497 ± 0.11303 92 2.0 hoursAfter IEC 4.51656 ± 0.03006 76 7.5 hoursTable 1 b: A620/A280 ratio Productivity % Estimation Time 2.59960 ± 0.24710 93 30.0 min. Step 8.0 hoursAfter concentration 4.40767 ± 0.03941 85After Sucrose gradientJournal of Research in Biology (2014) 3(8): 1132-1146 1136

El-Mohsnawy Eithar, 2014 Cell Destruction PC Purification Figure 1: Scheme shows different isolation and purification steps for phycocyanin purification. During the first purification step, two series of ammonium sulfate precipitation were applied.619 nm, respectively). It could be concluded that the membrane pellets exhibited no significant differencesextraction with HEPES buffer was the best kind of between phycocyanin extracted by HEPES buffer andextraction. Re-dissolving the thylakoid membrane in that extracted by HEPES buffer containing ß-DM,HEPES buffer not only enhanced the extraction of whereas a remarkable reduction was observed in thephycocyanin but also increased the amount of absorbance at 440 nm and 680 nm in case of extractionallophycocyanin. Absorption spectra of thylakoid by HEPES buffer only (Figure 3a). These results are1137 Journal of Research in Biology (2014) 3(8): 1132-1146

El-Mohsnawy Eithar, 2014 Absorbance (RU) 1.2 Abs orbanc e (R U) 1.2 HEPES extraction HEPES extraction ß-DM extraction 1 ß-DM extraction 1 Beads extraction Beads extraction Amm Sulf sediment 0.8 Amm Sulf sediment 0.8 0.6 0.6 0.4 0.4 0.2 0.2 00 A B250 350 450 550 650 750 450 500 550 600 650 700 Wa ve le ng th (nm ) Wa ve le ng th (nm ) Figure 2 : Absorption spectra of crude extracts by different conditions and after ammonium sulfate precipitation. 500 µl samples were measured by Shimadzu UV-2450 spectrophotometer. Absorption spectra 250-750 (A), absorbance 550-700 (B)Absorbance RU supported by 77K fluorescence spectra (Figure 3b), point to the presence of more allophycocyanin, PSII, and where a high peak was observed at 647 nm for both PSI in case of isolation by buffer containing ß-DM. isolation steps; whereas higher peaks were detected at 664 nm, 686 nm, and 733 nm for PSI. These spectra Purification 3 Thylak oid m em brane Ammonium sulfate precipitation P ellets after ß-DM ex trac tion Phycocyanin crude extract containing other 2.5 P ellets w ithout ß-DM ex trac tion impurities (allophycocyanin, photosystem complexes, and other soluble proteins) was exposed to two series of 2 ammonium sulfate precipitation. In the first step (20% ammonium sulfate), large hydrophobic proteins were 1.5 sedimented; whereas after the second step, phycocyanin 1 was precipitated. A remarkable reduction in the absorbance at 650 nm, 440 nm, and 280 nm (Figure 2a b) 0.5 was observed, which proves the high efficiency of these 0 two steps to remove most of the dissolved and large 250 350 450 550 650 750 hydrophobic contaminated proteins. These results were Wav ele ng th nm supported by A620/A280 value (3.494 ± 0.113) as shown in Table 1. This value is considered quite high, indicating Figure 3 a: Absorption spectra of pellets after the purity of phycocyanin. different extraction conditions. Pellets were suspended in HEPES 7.5 buffer till they reached an Although the absorption spectra and A620/A280 OD680 of 1.5−2. 500-µl samples were measured by a value pointed to pure phycocyanin, the emission Shimadzu UV-2450 spectrophotometer. fluorescence spectra showed the presence of some contamination (Figure 3b), where fluorescence emission Figure 3 b: 77K fluorescence emission spectra of spectra at 664 nm and 686 nm were detected apart from different extraction conditions compared with 647 nm, which indicates the presence of a few ammonium sulfate precipitation. Samples were contaminations of allophycocyanin in phycocyanin crude diluted with HEPES 7.5 buffer containing 60 % extracts. glycerol to OD620 = 0.05. The applied actinic light was 580 nm. Journal of Research in Biology (2014) 3(8): 1132-1146 1138

El-Mohsnawy Eithar, 2014Figure 4: Elution profile of purified phycocyanin dialysis against HEPES buffer for 8 hours. Changing ofusing IEC (Poros HQ/M). The column was dialysis buffer was done after 2 hours. POROS HQ/Mequilibrated by 8 CV of HEPES 7.5 buffer before column was equilibrated with HEPES buffer beforeloading. PC was eluted at 35 mM of MgSO4. loading partial purified phycocyanin. Figure 4 shows the elution gradient of MgSO4 (0-150 mM) with a step at 35Figure 5: Sucrose density gradient of concentrated mM that was used to elute highly purified phycocyanin.crude extract. 20% sucrose was frozen and slowly Pure phycocyanin was eluted at 35 mM of magnesiumthawed at 10 °C. 100 µl of OD620 nm 6 suspensions sulfate. Phycocyanin complex was desalted andwere slowly dropped onto the top of sucrose gradients concentrated to OD619 = 3. Quite a high A620/A280 valueand centrifuged at 36000 rpm for about 12 hours at (4.516 ± 0.03) was obtained.4°C (SW40-Rotor ultracentrifuge, Beckman).Second purification steps. Purification by sucrose density gradient Although the chromatographic purification Since purification by ammonium sulfateprecipitation did not reach an optimum A620/A280 value, presented a highly purified and large yield of C-C-phycocyanin extract needs an additional purification phycocyanin, sucrose gradient was found to be a fast andstep. A chromatographic step has been applied to reach effective step for the same purpose. Sucrose gradient wasan optimum value. prepared as described in the “Materials and Methods”Purification by IEC section. A highly contaminated crude extract-derived glass-bead extraction step was concentrated using a After 50% ammonium sulfate precipitation, the 10,000 Amicon tube before being dropped directly ontopellet was dissolved in HEPES buffer followed by the top surface of the sucrose gradient tube. After centrifugation, two distinct bands were observed. The lower one was C-phycocyanin, and the upper one was allophycocyanin (Figure 5). The phycocyanin band was collected, washed by HEPES buffer, and concentrated to OD619 = 3 before storing it. Phycocyanin evaluation of both methods Evaluation of the purification of C-phycocyanin did not stop at the level of A620/A280 values and total yield, whereas it extended to be investigated spectroscopically and by SDS-gel PAGE. Room temperature absorption spectra of C-phycocyanin purified by IEC and sucrose gradient exhibited almost the same behavior, where only one peak was detected at a maximum absorbance of 619 nm; whereas a reduction in the absorbance at 355 nm and 280 nm was observed. Moreover, the small shoulder at 650 nm disappeared. 77K emission fluorescence spectral investigations of phycocyanin purified by IEC or fractionated by sucrose gradient exhibited only one peak1139 Journal of Research in Biology (2014) 3(8): 1132-1146

El-Mohsnawy Eithar, 2014Figure 6 a: Absorption spectra of purified contamination. These results provided high evidence forphycocyanin after ammonium sulfate precipitation, the efficiency of the presented methods.IEC purification, and sucrose gradient. A partialpurified phycocyanin was used to visualize the A summary evaluation of chromatographic anddifference at 650 nm. 500-µl samples were measured sucrose gradient methods are shown in Tables 1a and 1b.by a Shimadzu UV-2450 spectrophotometer. There were no significant differences in A620/A280 values, whereas the total productivity was high in case of theFigure 6 b: 77K fluorescence emission spectra of sucrose gradient. In addition, a significant reduction inphycocyanin purified by ammonium sulfate purification time was observed in case of the sucroseprecipitation, IEC, and sucrose gradient, and these gradient.were precipitated by ammonium sulfate. Sampleswere diluted with HEPES 7.5 buffer containing 60 % DISCUSSIONglycerol to OD620 = 0.05. The applied actinic light was The extraction and the purification of580 nm. C-phycocyanin have been reported for differentat 647 nm; whereas shoulders at 664 nm and 686 nm cyanobacterial species using several steps. Thesedisappeared (Figure 6b). These results supported protocols required longer time and more equipment. Toabsorbance results and indicated the purity of the reach an optimum PC complex (large amount, pure, andcomplex. With regard to the A620/A280 value, purification in a short time), the production of C-phycocyanin passedby IEC and sucrose gradient produced 4.5 and 4.4 (Table through 2 main steps. The first step was the isolation of1a &b). These values pointed to high-quality C- PC, and the second one was purification. Each step wasphycocyanin. As shown in Figure 7, the SDS-gel monitored spectroscopically in order to achieve highelectrophoresis page, alpha, and beta phycocyanin efficiency.subunits are visualized without any additional Since the cyanobacterial cell wall is composed of peptidoglycan with an external lipopolysaccharide layer such as gram-negative bacteria, the design of cell destruction is very important, by which the cell wall is destroyed while keeping the thylakoid membrane in its native structure. As shown in the “Results” section, a combination of Lysozyme with 2000 psi was effective and mild. These results were in agreement with Gan et al., (2004) for Spirulina sp., Santos et al., (2004) for Calothrix sp., and Gupta and Sainis (2010) for Anacystis nidulans. The use of a combination of Lysozyme and glass beads was very strong and caused the destruction of both the cell wall and the thylakoid membrane, resulting in a huge amount of contamination, especially allophycocyanin. These contaminations extended to include photosystem complexes in case of using a buffer containing ß-DM. It should be pointed out that furtherJournal of Research in Biology (2014) 3(8): 1132-1146 1140

El-Mohsnawy Eithar, 2014Figure 7: SDS-gel PAGE of purified phycocyanin. extractions by HEPES buffer enhanced the isolation ofLane 1 marker protein, lane 2 phycocyanin purified the remaining C-phycocyanin, in addition to a largeby sucrose gradient and lane 3 phycocyanin purified amount of allophycocyanin. There was an inverseby IEC. relationship between the repetition of extraction and PC isolation, whereas a direct relationship has been recorded with regard to allophycocyanin (El-Mohsnawy, 2013). A model in Figure 8 illustrates a comparison between different isolation conditions. It could be concluded that a combination between Lysozyme and high pressure (2000 psi) with HEPES buffer was ideal for phycocyanin isolation with a low contamination. Different C-phycocyanin purification conditions have been widely investigated. A combination of two or more purification steps were usually applied till they reach a high A620/A280 ratio. A combination of ultrafiltration charcoal adsorption and spray drying was used to obtain C-PC with A620/A280 of 0.74 and a yield of 34%, whereas additional chromatographic steps were included to purify Components of crude extract Photosystem II Cytochrome b6f Allophycocyanin B-DM Glass-beads Photosystem I C-Phycocyanin Phospholipide ATPaseFigure 8: Model illustrates the major protein isolated as a result of different extraction conditions. This model is based on the results of absorbance and 77k fluorescence spectral analysis.1141 Journal of Research in Biology (2014) 3(8): 1132-1146

El-Mohsnawy Eithar, 2014C-PC to A620/A280 of 3.91 with a yield of 9% (Herrera optimization of each step. Several advantages of theet al., 1989). This method was improved by Gupta and sucrose gradient method are that it reduces the amount ofSainis (2010) and reached 2.18 and 4.72, respectively. lost PC complex during purification sequences, producesCombination of ammonium sulfate with a highly purified complex (A620/A280 value), and reduceschromatographic purification has been used for obtaining time; thus, it could be considered a standard model that isC-phycocyanin in different purity levels and applied in different cyanobacteria species and too simplerecommended by Rito-Palomares et al., 2001 and Song not to be used by specialists.et al., 2013. On the other hand, the use of two-phaseaqueous extraction followed by chromatographic ACKNOWLEDGMENTSpurification was recently reported by Soni et al., 2008. I would like to express my deep thanks forAlthough it produced extremely pure C-phycocyaninwith A620/A280=6.69, the total yield was affected. In the Prof. Rögner Matthias (Ruhr University Bochum), whopresent work, two strategies have been applied. The first giving me the opportunity to measure someone was based on two steps: ammonium sulfate chromatographic and spectroscopical measurements.precipitation followed by chromatographic purification Also, I acknowledge the German Research Council, DFG(IEC). The second strategy was based on the for the financial support. Prof. Kurisu, Genji (Proteinconcentration of crude extract followed by sucrose Center of Osaka University) is gratefully acknowledgedgradient fractionation. Through concentration of crude for permitting me to do a part of practical work inextract was considered important not only for his laboratory. I would like to thank Hisako Kubota forconcentration C-phycocyanin but also for the removal of fruit discussions. I would like to thank Mrs Reginathe small-molecular-weight soluble protein. Oworah-Nkruma for technical assistance rendered. To evaluate this new purification step (sucrose REFERENCESgradient), a highly contaminated PC crude extract Adir N, Dobrovetsky Y and Lerner N. 2001. Structure(Lysozyme with glass beads) was concentrated by an of c-phycocyanin from the thermophilic cyanobacteriumAmicon 10,000 centrifugation tube and exposed to Synechococcus vulcanus at 2.5 Å: structural implicationssucrose density gradient fractionation. The astonishing for thermal stability in phycobilisome Assembly. J Molresults were recorded by the sucrose gradient that gave Biol. 313(1):71–81.almost the same purity and a much better yield. Adir N. 2005. Elucidation of the molecular structures of After several optimization sequences, it could be components of the phycobilisome: reconstructing a giant.recommended that the digestion of T. elongatus cell wall Photosynth. Res., 85(1): 15–32.by Lysozyme and the exposure to high pressure (2000psi) followed by PC extraction by HEPES buffer once or Bennett A and Bogorad L. 1973. Complementarytwice was found to be the best condition for the isolation chromatic adaptation in a filamentous blue-green alga.of partial pure PC. This crude extract should be J Cell Biol., 58(2):419-35.concentrated through an Amicon 10,000 centrifugationtube before fractionation by the sucrose gradient.Isolation and purification should be quick, reliable, andefficient; so, absorbance and fluorescence spectrafacilitated the purity of C-phycocyanin, thus enabling theJournal of Research in Biology (2014) 3(8): 1132-1146 1142

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